Abstract The primary cause of death in patients with acute lymphoblastic leukemia (ALL) is sudden disease relapse (80% of patients with minimum residual disease, MRD, will relapse). For better outcome, onset of relapse must be detected early. Therefore, frequent monitoring of MRD is needed. In pediatric ALL, monitoring MRD utilizes multi- parameter flow cytometry (MFC) or qPCR from bone marrow aspirates (BMA), which is painful and an invasive procedure, hence not performed frequently. MFC/BMA detects MRD in ~40% of patients; most patients are classified as MRD negative in spite of residual cancer cells present in their blood. qPCR detection in B-ALL is labor intensive/expensive as it requires patient-specific target identification via sequencing. In some B-ALL patients, specific DNA markers cannot be identified. Although MRD status has proven to be an effective prognostic indicator allowing for risk assessment and improving survival with treatment intensification, MFC and qPCR require a BMA due to limited ability to detect rare leukemia cells in peripheral blood. Bone marrow is not the only niche where leukemia cells are found. Circulating leukemia cells (CLCs) are present in patient?s peripheral blood. Therefore, if sensitive methods for MRD detection from blood could be used, frequent MRD monitoring could improve patient outcome. Built from a successful R21 IMAT project for MRD monitoring in adult acute myeloid leukemia (AML) patients, an innovative and highly sensitive MRD platform will be delivered herein, which will consist of a fluidic cartridge that isolates and analyzes enriched CLCs from peripheral blood in an automated fashion. The cartridge is comprised of two modules, one for the affinity selection of B- or T-type CLCs from blood and the other module used to ?trap? enriched cells to allow for immunophenotyping and/or FISH analyses (FISH performed on high-risk patients). In this R33 application, the utility of this microfluidic assay will be demonstrated in pediatric B-ALL and T-ALL patients. Appropriate antibodies (anti-CD19 and anti-CD7 monoclonal antibodies for B-ALL and T-ALL, respectively) will be immobilized to a surface of the cell selection module with the aid of a photocleavable linker to affinity-select cells expressing CD19 or CD7 surface antigens. Cells will be photolytically released from the capture surface and CLCs identified by the expression of aberrant markers, such as Terminal deoxynucleotidyl Transferase (TdT), in a unique microtrap (TRAP) module. This module allows for automated cell staining and phenotyping. The CLC microfluidic cartridge will be clinically validated by monitoring MRD status in B-ALL and T-ALL pediatric patients and CLC burden will be tracked. The cartridge will perform CLC FISH analysis for high-risk patients to gain information on chromosomal aberrations that provide information to allow precision treatment. Given the strong data generated to date in AML (100% test positivity) and the urgent diagnostic need for an improved easy-to-implement MRD assay that permits frequent sampling using peripheral blood, broad patient coverage, and early detection of relapse, the proposed cartridge will fill an unmet clinical need in pediatric oncology as well as other leukemic diseases.